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Journal of Clinical Microbiology, October 2001, p. 3812-3813, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3812-3813.2001
LETTERS TO THE EDITOR
Whole-Blood Hepatitis C Virus RNA Extraction Methods
 |
LETTER |
We read the article by Cook et al. (3) with
interest, since they compared their findings with our previous work
(4-8; D. Brashear, S. Taylor, J. Xiang, D. Klinzman, F. LaBrecque, M. Pfaller, B. Alden, D. LaBrecque, J. Stapleton, and W. Schmidt, 10th Triennial Int. Symp. Viral Hepatitis
Liver Dis., abstr. C008, 2000; J. Xiang, F. LaBrecque, W. N. Schmidt, D. Klinzman, D. Brashear, D. R. LaBrecque, M. J. Perino-Phillips, and J. T. Stapleton, 10th Triennial Int. Symp.
Viral Hepatitis Liver Dis., abstr. F011, 2000). Cook et al. did
not find that whole blood was a more sensitive source of hepatitis C
virus (HCV) RNA than plasma. Careful review of their paper revealed
several potential reasons for this discrepancy, including the
following. (i) A different method of extracting RNA from whole blood
was used (3). We did not use Trizol following extraction
with Catrimox (4, 9). The pH of the phenol-guanidinium mixture in Trizol is not published. We found that the phenol pH is
critical for optimal extraction (4, Brashear et al.,
10th Triennial Int. Symp. Viral Hepatitis Liver Dis.). In addition, we
showed that extraction with Trizol was less sensitive than our Catrimox
method (Xiang et al., 10th Triennial Int. Symp. Viral Hepatitis Liver
Dis.). (ii) Cook et al. added carrier RNA to serum but not to whole
blood (3), yet we have shown that the addition of carrier
RNA to either plasma or whole blood increases sensitivity (Brashear et
al., 10th Triennial Int. Symp. Viral Hepatitis Liver Dis.). (iii) Cook
et al. used different primers and thermocycler conditions than did we,
and they used only single-round reverse transcription (RT)-PCR instead
of nested RT-PCR (3, 4). (iv) Finally, the serum volume
was not adjusted to the whole-blood volume (3), which we
took into account (6).
As suggested by Cook et al., intracellular HCV RNA is a potential
explanation for the improved sensitivity of HCV RNA detection in whole
blood (3), yet we found that a significant portion of HCV
RNA in the crude cellular pellet was not intracellular (6). Our study of 115 patients with HCV infection
demonstrated that the improved sensitivity of whole-blood HCV RNA
detection correlated with the presence and quantity of cryoglobulins
(8). We also noted highly significant differences in the
detection of HCV RNA for whole blood and plasma among 52 interferon-treated patients who were monitored throughout treatment
(7). As was the case for the one patient studied by Cook
et al. (3), among our 52 patients the clearance of
intracellular virus occurred nearly simultaneously with the clearance
of plasma virus during therapy (7).
The extraneous bands described by the authors (3) are not
routinely present in our work (e.g., see Fig. 1, 3, and 5B in reference
4). In addition, Cook et al. stated that it was not tenable that our antibody-negative subjects were truly infected with
HCV since we did not evaluate liver biopsy samples for HCV RNA
(3). However, we proved that several HCV antibody-negative patients had HCV RNA present in multiple whole-blood and plasma samples
collected over months and years, using primers from several different
regions of the genome (4, 6). In addition, we confirmed
our results by Southern blotting and sequence analysis of PCR products
and by direct comparison with results obtained with the use of
commercial HCV RNA assays (4, 6, 9). The finding of
prolonged RNA positivity in the absence of detectable HCV antibody has
been described by others (2), including after the
experimental infection of chimpanzees (1). In conclusion, we believe that the difference between our work and that of Cook et al.
(3) relates to differences in the whole-blood RNA
extraction methodology.
| |
REFERENCES |
| 1.
|
Bassett, S. E.,
K. M. Brasky, and R. E. Lanford.
1998.
Analysis of hepatitis C virus-inoculated chimpanzees reveals unexpected clinical profiles.
J. Virol.
72:2589-2599[Abstract/Free Full Text].
|
| 2.
|
Bonacini, M., and M. Puoti.
2000.
Hepatitis C in Patients With Human Immunodeficiency Virus Infection Diagnosis, Natural History, Meta-analysis of Sexual and Vertical Transmission, and Therapeutic Issues.
Arch. Intern. Med.
160:3365-3373[Abstract/Free Full Text].
|
| 3.
|
Cook, L.,
A. M. Ross,
G. B. Knight, and V. Agnello.
2001.
Use of whole blood specimens for routine clinical quantitation of hepatitis C virus RNA does not increase assay sensitivity.
J. Clin. Microbiol.
38:4326-4331[Abstract/Free Full Text].
|
| 4.
|
Schmidt, W. N.,
D. Klinzman,
D. LaBrecque,
D. E. Macfarlane, and J. T. Stapleton.
1995.
Direct detection of hepatitis C virus (HCV) RNA from whole blood, and comparison with HCV RNA in plasma and peripheral blood mononuclear cells.
J. Med. Virol.
47:153-160[Medline].
|
| 5.
|
Schmidt, W. N.,
P. Wu,
J. Cederna,
F. A. Mitros,
D. R. LaBrecque, and J. T. Stapleton.
1997.
Surreptitious hepatitis C virus (HCV) infection detected in the majority of patients with cryptogenic chronic hepatitis and negative HCV antibody tests.
J. Infect. Dis.
176:27-33[Medline].
|
| 6.
|
Schmidt, W. N.,
P. Wu,
J.-Q. Han,
M. J. Perino,
D. R. LaBrecque, and J. T. Stapleton.
1997.
Distribution of hepatitis C virus (HCV) RNA in whole blood and blood cell fractions: plasma HCV RNA analysis underestimates circulating virus load.
J. Infect. Dis.
176:20-26[Medline].
|
| 7.
|
Schmidt, W. N.,
P. Wu,
D. Brashear,
D. Klinzman,
M. J. Perino-Phillips,
D. R. LaBrecque, and J. T. Stapleton.
1998.
Effect of interferon therapy on hepatitis C virus RNA in whole blood, plasma and peripheral blood mononuclear cells.
Hepatology
28:1110-1116[CrossRef][Medline].
|
| 8.
|
Schmidt, W. N.,
J. T. Stapleton,
D. R. LaBrecque,
F. A. Mitros,
K. Kirkegaard,
M. J. P. Phillips, and D. Brashear.
2000.
Hepatitis C infection and cryoglobulinemia: analysis of whole blood and plasma HCV RNA concentration and correlation with liver histology.
Hepatology
31:737-744[CrossRef][Medline].
|
| 9.
|
Stapleton, J. T.,
D. Klinzman,
W. N. Schmidt,
P. Wu,
D. R. LaBrecque,
J.-Q. Han,
M. J. Perino-Phillips,
R. Woolson, and B. Alden.
1999.
Prospective comparison of whole blood and plasma hepatitis C virus RNA detection systems: Improved detection using whole blood as the source of viral RNA.
J. Clin. Microbiol.
37:484-489[Abstract/Free Full Text].
|
| | | | |
Warren Schmidt
Division of Hepatology
The University of Iowa
Iowa City, Iowa
|
| | | | |
Jack T. Stapleton
Division of Infectious Diseases
The University of Iowa
Iowa City, Iowa
|
| |
AUTHORS' REPLY |
Drs. Schmidt and Stapleton think that the difference between our work
(3) and theirs (6) is related to
differences in whole-blood RNA extraction methods due to a presumed
difference in pH of the phenol-guanidinium. The Trizol U.S. patent
documents state that the pH of Trizol is 4.0, the exact pH used in
their method (D. Brashear, S. Taylor, J. Xiang, D. Klinzman, F. LaBrecque, M. Pfaller, B. Alden, D. LaBrecque, J. Stapleton, and W. Schmidt, 10th Triennial Int. Symp. Viral Hepatitis Liver Dis., abstr.
C008, 2000). Therefore, Trizol and the acid phenol-guanidinium mixture used by Schmidt and Stapleton are equivalent chemical environments and
the fact that our data failed to confirm their findings is not
explained by differences in extraction methods. Nor are the differences
between PCR methodology, carrier RNA, and volume adjustment consequential. The competitive reverse transcription (RT)-PCR method we
employed was quantitative, whereas a semiquantitative method was
employed by Schmidt and Stapleton. Sufficient cellular RNA is present
in the whole-blood samples so that the addition of carrier RNA does not
enhance the amount of hepatitis C virus (HCV) RNA isolated. We did not
physically adjust the serum volume to the whole-blood volume, but when
adjustment was made in the calculations (see Table 2 of reference
3), there was only a slight difference in the
correlation between the serum and whole-blood results.
Extraneous bands are quite apparent in Fig. 3 and 5 of reference
6. Despite differences in the primers used in the
two studies, spurious amplification appears to have occurred with both
methods. As demonstrated by the sequences we detected, these extraneous
bands are generated from false priming and artifactual amplification of
human cellular RNA at high concentrations, when HCV RNA is absent; this
cannot be circumvented entirely by increasing the stringency of the
RT-PCR.
The finding of extracellular HCV RNA in the crude cellular pellet could
explain differences in HCV quantification when using whole blood or
serum but only when the blood or serum was stored at 4°C prior
to separation as in the Schmidt et al. study (4). In
routine clinical assays, the serum or plasma is not refrigerated prior
to separation. Schmidt and Stapleton are correct: cryoglobulins, particularly type II cryoglobulins, can be responsible for loss of
virus. Their study on cryoglobulins (5) confirms the
original observation that HCV can be precipitated with cryoglobulins
(1) but does not provide a rationale for routine clinical
use of their method. The cryoglobulins associated HCV infection
(2) are not the type II cryoglobulins that precipitate at
room temperature. Hence, for routine specimens the current methodology
is adequate. For patients with type II cryoglobulinemia, blood should
be clotted at 37°C prior to processing.
We reiterate that the presence of HCV in blood cells without the
demonstration of HCV RNA in the liver is untenable. If there were
replication of HCV in blood cells, an idea that is controversial and
therefore requires verification, then there must be replication in the
liver. A verification of finding HCV in the blood cells but not in the
serum of seronegative patients is detection of HCV RNA in the liver.
An improved method for detection of HCV RNA in blood samples would be
welcomed in diagnostic clinical laboratories; unfortunately, we did not
find the that the Schmidt-Stapleton methodology provided any
improvement over our current method.
| |
REFERENCES |
| 1.
|
Agnello, V.,
R. T. Chung, and L. M. Kaplan.
1992.
A role for hepatitis C virus infection in type II cryoglobulinemia.
N. Engl. J. Med.
327:1490-1495[Abstract].
|
| 2.
|
Agnello, V.
1998.
Mixed cryoglobulinemia after hepatitis C virus: more or less ambiguity.
Ann. Rheum. Dis.
57:701-702[Free Full Text].
|
| 3.
|
Cook, L.,
A. M. Ross,
G. B. Knight, and V. Agnello.
2001.
Use of whole blood specimens for routine clinical quantitation of hepatitis C virus RNA does not increase assay sensitivity.
J. Clin. Microbiol.
38:4326-4331.
|
| 4.
|
Schmidt, W. N.,
P. Wu,
J.-Q. Han,
M. J. Perino,
D. R. Labrecque, and J. T. Stapleton.
1997.
Distribution of hepatitis C virus (HCV) RNA in whole and blood cell fractions: Plasma HCV RNA analysis underestimates circulating virus load.
J. Infect. Dis.
176:20-26.
|
| 5.
|
Schmidt, W. N.,
J. T. Stapleton,
D. R. Labrecque,
F. A. Mitros,
K. Kirkegaard,
M. J. P. Phillips, and D. Brashear.
2000.
Hepatitis C infection and cryoglobulinimia: analysis of whole blood and plasma HCV RNA concentration and correlation with liver histology.
Hepatology
31:737-744.
|
| 6.
|
Stapleton, J. T.,
D. Klinzman,
W. N. Schmidt,
P. Wu,
D. R. LaBrecque,
J.-Q. Han,
M. J. Perino-Phillips,
R. Woolson, and B. Alden.
1999.
Prospective comparison of whole blood and plasma hepatitis C virus RNA detection systems: improved detection using whole blood as the source of viral RNA.
J. Clin. Microbiol.
37:484-489.
|
| | | | |
Glenn B. Knight
Vincent Agnello
Lahey Clinic
Burlington, Massachusetts
|
Journal of Clinical Microbiology, October 2001, p. 3812-3813, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3812-3813.2001
